Among many analytical methods of detecting and quantifying nucleic acids, PCR is one of the most commonly used methods, the principles of which are disclosed in U.S. Pat. Nos. 4,683,195 and 4,683,202. Despite the obvious advantages of the PCR methodology, the ability to amplify low copy DNA template sequences by several orders of magnitude also means that the success of a PCR amplification reaction is determined in large part already during the first PCR cycle, where non-specific priming can result in the amplification of spurious PCR products, such as primer dimers. Once present in the reaction, these non-specific PCR products are amplified along with the targeted DNA sequence thereby compromising the specificity and the overall efficiency of a PCR amplification reaction.
In an effort to suppress non-specific priming, so-called ‘hot start’ PCR protocols, have been devised in which the polymerase activity is suppressed prior to the initial denaturation of the template and PCR primers during the first PCR cycle. This approach therefore seeks to minimize spurious primer extension at low temperatures that can lead to the formation of primer dimers or other non-specific PCR products. Suppressing polymerase activity at low temperatures is however problematic when the PCR primers themselves anneal specifically to the targeted DNA sequence at low temperatures.
For the foregoing reasons, there is an unmet need in the art for improved hot-start PCR protocols.